Low GNG12 Expression Predicts Adverse Outcomes: A Potential Therapeutic Target for Osteosarcoma.

Background: G protein subunit gamma 12 (GNG12) is observed in some types of cancer, but its role in osteosarcoma is unknown. This study hypothesized that GNG12 may be a potential biomarker and therapeutic target. We aimed to identify an association between GNG12 and osteosarcoma based on the Gene Expression Omnibus and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) databases. Methods: Osteosarcoma samples in GSE42352 and TARGET database were selected as the test cohorts. As the external validation cohort, 78 osteosarcoma specimens from The Second Affiliated Hospital of Nanchang University were collected. Patients with osteosarcoma were divided into high and low GNG12 mRNA-expression groups; differentially expressed genes were identified as GNG12-related genes. The biological function of GNG12 was annotated using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, gene set enrichment analysis, and immune infiltration analysis. Gene expression correlation analysis and competing endogenous RNA regulatory network construction were used to determine potential biological regulatory relationships of GNG12. Overall survival, Kaplan-Meier analysis, and log-rank tests were calculated to determine GNG12 reliability in predicting survival prognosis. Results: GNG12 expression decreased in osteosarcoma samples. GNG12 was a highly effective biomarker for osteosarcoma [area under the receiver operating characteristic (ROC) curve (AUC) = 0.920], and the results of our Kaplan-Meier analysis indicated that overall survival and progression-free survival differed significantly between low and high GNG-expression group (p < 0.05). Functional analyses indicated that GNG12 may promote osteosarcoma through regulating the endoplasmic reticulum. Expression correlation analysis and competing endogenous RNA network construction showed that HOTTIP/miR-27a-3p may regulate GNG12 expression. Furthermore, the subunit suppresses adaptive immunity via inhibiting M1 and M2 macrophage infiltration. GNG12 was inhibited in metastatic osteosarcoma compared with non-metastatic osteosarcoma, and its expression predicted survival of patients (1, 3, and 5-year AUCs were 0.961, 0.826, and 0.808, respectively). Conclusion: This study identified GNG12 as a potential biomarker for osteosarcoma prognosis, highlighting its potential as an immunotherapy target.

Profiling of gut microbial dysbiosis in adults with myeloid leukemia.

Dysregulation of gut microbiota is implicated in the pathogenesis of various diseases, including metabolic diseases, inflammatory diseases, and cancer. To date, the link between gut microbiota and myeloid leukemia (ML) remains largely unelucidated. Herein, a total of 29 patients with acute myeloid leukemia (AML), 17 patients with chronic myeloid leukemia (CML), and 33 healthy subjects were enrolled, and gut microbiota were profiled via Illumina sequencing of the 16S rRNA. We evaluated the correlation between ML and gut microbiota. The microbial α-diversity and ß-diversity exhibited significant differences between ML patients and healthy controls (HCs). Compared to healthy subjects, we found that at the phylum level, the relative abundance of Actinobacteria, Acidobacteria, and Chloroflexi was increased, while that of Tenericutes was decreased. Correspondingly, at the genus level in ML, Streptococcus were increased, especially in AML patients, while Megamonas (P = 0.02), Lachnospiraceae NC2004 group, and Prevotella 9 (P = 0.007) were decreased. Moreover, ML-enriched species, including Sphingomonas, Lysobacyer, Helicobacter, Lactobacillus, Enterococcus, and Clostridium sensu stricto 1, were identified. Our results indicate that the gut microbiota was altered in ML patients compared to that of healthy subjects, which could contribute to the elucidation of microbiota-related pathogenesis of ML, and the development of novel therapeutic strategies in the treatment of ML.

miR-384-5p regulates inflammation in Candida albicans-induced acute lung injury by downregulating PGC1ß and enhancing the activation of Candida albicans-triggered signaling pathways.

Acute lung injury (ALI) is one of the most prevalent respiratory syndromes of excessive inflammatory reaction during lung infection. Candida albicans (C. albicans) infection is among the leading causes of ALI. MicroRNAs (miRNAs) regulate the expression of target mRNAs, including those involved in inflammatory processes, by binding to the 3'UTR. To date, the roles of miRNAs in C. albicans-induced ALI remain unclear. In this study, we investigated the role of miR-384-5p in C. albicans-induced ALI and its underlying molecular mechanism. RT-PCR, Western blot, ELISA, Myeloperoxidase (MPO) assay, microRNA target analysis, transient transfection, and luciferase reporter assay were utilized. In vivo study was conducted on mouse model. The expression of miR-384-5p was upregulated and positively correlated with inflammatory cytokine production in lung tissues and RAW264.7 and J774A.1 macrophages infected with C. albicans. The miR-384-5p inhibitor alleviated the inflammatory reaction induced by C. albicans. Target prediction analysis revealed that PGC1ß was a target of miR-384-5p, which was further validated by the PGC1ß 3'-UTR luciferase assay and the inverse correlation between the expression of miR-384-5p and PGC1ß in C. albicans-infected ALI tissues and macrophages. Moreover, macrophages transfected with miR-384-5p mimic exhibited reduced levels of PGC1ß. The suppression of the expression of PGC1ß by C. albicans infection in the macrophages was abrogated by miR-384-5p inhibitor. Then, we demonstrated that PGC1ß played an inhibitory role in C. albicans-induced production of inflammatory cytokines. Furthermore, suppression of miR-384-5p in macrophages inhibited the activation of the NF-κB, MAPK, and Akt signaling pathways triggered by C. albicans, but not the STAT3 pathway. These results demonstrate that miR-384-5p contributes to C. albicans-induced ALI at least in part by targeting PGC1ß and enhancing the activation of the NF-κB, MAPK, and Akt inflammatory signaling pathways. Thus, targeting miR-384-5p might exert a protective effect on C. albicans-induced ALI.

Increased Th17 cells and IL-17A exist in patients with B cell acute lymphoblastic leukemia and promote proliferation and resistance to daunorubicin through activation of Akt signaling.

BACKGROUND: Immune regulation is crucial for the pathogenesis of B-cell acute lymphoblastic leukemia (B-ALL). It has been reported that Th17 cells as a newly identified subset of CD4(+) T cells are involved in the pathogenesis of several hematological disorders. However, the role of Th17 cells in the pathophysiology of B-ALL is still unclear. METHODS: The frequencies of T cells were determined by flow cytometry in the peripheral blood and bone marrow of 44 newly diagnosed B-ALL patients and 25 age-matched healthy donors. The cell viability and apoptosis were determined by CCK-8 assay and Annexin V staining, respectively. Western blot was applied to identify the level of Akt and Stat3 phosphorylation. RESULTS: We assessed and observed a significantly increased frequency of Th17 cells and a drastically decreased frequency of Th1 cells in peripheral blood mononuclear cells and bone marrow mononuclear cells from newly diagnosed B-ALL patients compared with healthy donors. Furthermore, increased levels of Th17-related cytokines including IL-17, IL-21, IL-23, IL-1ß, and IL-6 were presented in between blood and marrow in B-ALL patients. Both IL-17A and IL-21, two Th17-secreted cytokines, induced the proliferation of B-ALL cell line Nalm-6 and patient B-ALL cells isolated from B-ALL patients, herein either cytokine led to the phosphorylation of Akt and Stat3. Additionally, IL-17A promoted resistance to daunorubicin via activation of Akt signaling and the PI3K/Akt inhibitor LY294002 or perifosine almost completely rescued daunorubicin-induced cell death in B-ALL cells. CONCLUSIONS: Our findings suggest that elevated Th17 cells secrete IL-17A by which promotes the proliferation and resistance to daunorubicin in B-ALL cells through activation of Akt signaling. Th17 cells may represent a novel target to improve B-ALL immunotherapy.

Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia and Enhances Chemosensitivity to Daunorubicin.

RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.

Epigenetic deregulated miR-375 contributes to the constitutive activation of JAK2/STAT signaling in myeloproliferative neoplasm.

Constitutive activation of Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT) signaling caused by JAK2V617F and other mutations is central to the pathogenesis of myeloproliferative neoplasm (MPN). Negative regulators such as suppressors of cytokine signaling (SOCS) inhibit activated JAK2/STAT signaling. However, whether silencing of negative regulators facilitates JAK2/STAT signaling is unclear. Here, we report that loss of miR-375 expression contributes to the constitutive activation of JAK2/STAT signaling. MiR-375 reduced JAK2 protein level and repressed the activity of a luciferase reporter by binding 3'-untranslated regions, which was abolished by the mutation of the predicted miR-375-binding site. Meanwhile, a significant inverse correlation between the expressions of miR-375 and JAK2 was found in multiple types of leukemic cell lines and bone marrow mononuclear cells from MPN patients, suggesting that JAK2 may be a miR-375 target gene. Furthermore, forced expression of miR-375 inhibited constitutive and inducible JAK2/STAT signaling, suppressed cell proliferation, and decreased colony formation in hematopoietic progenitors from MPN patients. Finally, histone deacetylation (HDAC) inhibitors restored miR-375 expression, which was much lower in patients with MPN compared with healthy volunteers. Collectively, our data suggest that the loss of miR-375 expression enhances the constitutive and persistent activation of JAK2/STAT signaling. Restoration of miR-375 expression might contribute to the clinical treatment for MPN patients.

[Expression of musashi-2 gene in leukemia stem cells from acute myeloid leukemia patients].

This study was aimed to detect the expression of Musashi-2 (Msi2) in acute myeloid leukemia (AML) and investigate the relationship between Msi2 and other clinical parameters, especially CD34. A total RNA was extracted from bone marrow of newly diagnosed AML patietns. The Msi2 mRNA expression in newly diagnosed AML patients was detected with real-time fluorescence quantitative RT-PCR. The expression level of CD34 in above-menthioned patients was detected by flow cytometry (FCM). The relationship between the expression of Msi2 mRNA and clinical outcome in AML patients was analysed. The results showed that (1)the expression of Msi2 mRNA in newly diagnosed AML patients was much higher than that in healthy volunteers (P < 0.05) , especially in M1, M4 and M5 patients; (2)the expression level of Msi2 did not correlate with age, sex, white blood cell count of peripheral blood, AML1/ETO and PML/RARa fusion gene (P > 0.05); (3) Msi2 expression level in patients with CD34(+) cells was significantly higher than that in patients with CD34(-) cells (P < 0.05). It is concluded that the Msi2 mRNA expresses in leukamia stem cells, the high expression of Msi2 mRNA has been found in newly diagnosed AML patients, especially in M1, M4 and M5 patients, the high expression also has been observed in patients with CD34(+).

Th17 cells and interleukin-17 increase with poor prognosis in patients with acute myeloid leukemia.

Although Th17 cells play crucial roles in the pathogenesis of many autoimmune and inflammatory disorders, their roles in malignancies are currently under debate. The role and mechanism of Th17 cells in patients with acute myeloid leukemia (AML) remain poorly understood. Here we demonstrated that the frequency of Th17 cells was significantly increased in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells from AML patients compared with healthy donors. Plasma levels of interleukin (IL)-17, IL-22, IL-23, IL-1ß, IL-6, and transforming growth factor (TGF)-ß1 were significantly increased in blood and bone marrow in AML patients compared with healthy donors. The in vitro experiments demonstrated that IL-1ß, IL-6, IL-23, but not TGF-ß1 promoted the generation and differentiation of Th17 cells from naive CD4(+) T cells in humans. IL-17A, a signature cytokine secreted by Th17 cells, induced the proliferation of IL-17 receptor (IL-17R)-positive AML cells via IL-17R, in which activation of PI3K/Akt and Jak/Stat3 signaling pathway may play important roles. In addition, combination of IL-17A and IL-22 significantly reduced the generation of Th1 cells and the production of interferon (IFN)-γ from healthy donor or AML patient peripheral blood mononuclear cells. Patients with high Th17 cell frequency had poor prognosis, whereas patients with high Th1 cell frequency had prolonged survival. Combined analysis of Th1 and Th17 cell frequencies improved the ability to predict patient outcomes. In conclusion, Th17 cells play a crucial role in the pathogenesis of AML and may be an important therapeutic target and prognostic predictor.

Anticancer effects of baicalein on hepatocellular carcinoma cells.

The therapeutic potential of baicalein against hepatoma cells was evaluated in vitro and in vivo. In cell viability assays, baicalein showed significant cytotoxicity against the hepatocellular carcinoma cell lines H22, Bel-7404, and Hep G2 and moderate cytotoxicity against immortalized human hepatocytes. Baicalein induced G0/G1-phase arrest in hepatocellular carcinoma cells, inhibited AKT, and promoted the degradation of ß-catenin and cyclin D1 without activation of GSK-3ß. Furthermore, baicalein significantly inhibited H22 xenograft tumor growth without causing obvious adverse effects on weight or liver and spleen weight indexes in ICR mice. Immunohistochemical analysis showed that the inhibition of tumor growth in baicalein-treated mice was associated with decreased AKT, ß-catenin, and cyclin D1 expression ex vivo. Our data indicate that baicalein might regulate cyclin D1 transcription via a ß-catenin-dependent mechanism, leading to cell cycle arrest at G0/G1 phase and impaired cancer cell proliferation. These results suggest that baicalein is a potential candidate for the treatment of hepatocellular carcinoma.

[In vitro study on injectable alginate-strontium hydrogel for bone tissue engineering].

OBJECTIVE: To investigate the application potential of alginate-strontium (Sr) hydrogel as an injectable scaffold material in bone tissue engineering. METHODS: The alginate-Sr/-calcium (Ca) hydrogel beads were fabricated by adding 2.0 wt% alginate sodium to 0.2 mol/L SrCl2/CaCl2 solution dropwise. Microstructure, modulus of compression, swelling rate, and degradability of alginate-Sr/-Ca hydrogels were tested. Bone marrow mesenchymal stem cells (BMSCs) were isolated from femoral bones of rabbits by flushing of marrow cavity. BMSCs at passage 5 were seeded onto the alginate-Sr hydrogel (experimental group) and alginate-Ca hydrogel (control group), and the viability and proliferation of BMSCs in 2 alginate hydrogels were assessed. The osteogenic differentiation of cells embeded in 2 alginate hydrogels was evaluated by alkaline phosphate (ALP) activity, osteoblast specific gene [Osterix (OSX), collagen type I, and Runx2] expression level and calcium deposition by fluorescent quantitative RT-PCR and alizarin red staining, Von Kossa staining. The BMSCs which were embeded in alginate-Ca hydrogel and cultured with common growth medium were harvested as blank control group. RESULTS: The micromorphology of alginate-Sr hydrogel was similar to that of the alginate-Ca hydrogel, with homogeneous pore structure; the modulus of compression of alginate-Sr hydrogel and alginate-Ca hydrogel was (186.53 +/- 8.37) and (152.14 +/- 7.45) kPa respectively, showing significant difference (t=6.853, P=0.002); there was no significant difference (t=0.737, P=0.502) in swelling rate between alginate-Sr hydrogel (14.32% +/- 1.53%) and alginate-Ca hydrogel (15.25% +/- 1.64%). The degradabilities of 2 alginate hydrogels were good; the degradation rate of alginate-Sr hydrogel was significantly lower than that of alginate-Ca hydrogel on the 20th, 25th, and 30th days (P < 0.05). At 1-4 days, the morphology of cells on 2 alginate hydrogels was spherical and then the shape was spindle or stellate. When three-dimensional cultured for 21 days, the DNA content of BMSCs in experimental group [(4.38 +/- 0.24) g] was significantly higher than that in control group [(3.25 +/- 0.21) g] (t=8.108, P=0.001). On the 12th day after osteogenic differentiation, the ALP activity in experimental group was (15.28 +/- 1.26) U/L, which was significantly higher than that in control group [(12.07 +/- 1.12) U/L] (P < 0.05). Likewise, the mRNA expressions of OSX, collagen type I, and Runx2 in experimental group were significantly higher than those in control group (P < 0.05). On the 21th day after osteogenic differentiation, alizarin red staining and Von Kossa staining showed calcium deposition in 2 groups; the calcium nodules and phosphate deposition in experimental group were significantly higher than those in control group (P < 0.05). CONCLUSION: Alginate-Sr hydrogel has good physicochemical properties and can promote the proliferation and osteogenic differentiation of BMSCs, so it is an excellent injectable scaffold material for bone tissue engineering.

Synergistic apoptosis induction in leukemic cells by miR-15a/16-1 and arsenic trioxide.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate target gene expression through translation repression or messenger RNA degradation. MiR-15a and 16-1 form a cluster at the chromosomal region 13q14, which is frequently deleted or down-regulated in chronic lymphocytic leukemia. Arsenic trioxide (As(2)O(3), ATO) has been successfully applied to treat acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in hematologic and solid tumors. Here we investigated the potential synergy between miR-15a/16-1 and ATO on Bcr-Abl positive leukemic K562 cells. In this study, we found that combination of miR-15a/16-1 and ATO synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. However, the expression of Bcr-Abl was not affected by ATO and/or miR-15a/16-1. Moreover, apoptotic synergy between miR-15a/16-1 and ATO was observed in Bcr-Abl negative leukemic cell lines and primary leukemic cells. Taken together, these findings suggested that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of leukemia.

The conformation change of Bcl-2 is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in SGC7901 human gastric cancer cells.

BACKGROUND: Arsenic trioxide has been established as a first-line agent for treating acute promyelocytic leukemia. Experimental data suggest that arsenic trioxide also can have a potential use as chemotherapeutic agent for other malignancies. The precise mechanisms of action of arsenic trioxide have though not been elucidated. As the role of Bcl-2 in arsenic trioxide-mediated cell apoptosis and conformation change of Bcl-2 in response to arsenic trioxide treatment has not been studied. The aim of the present study was to determine whether conformation change of Bcl-2 is involved in the action of arsenic trioxide. METHODS: Human gastric cancer SGC7901 cells were exposed to different concentrations of arsenic trioxide. Proliferation was measured by using the Kit-8 cell counting assay. Analysis of nuclear morphology was observed by DAPI staining. The apoptosis rates of cells treated with arsenic trioxide were analyzed by flow cytometry using Annexin V-FITC staining. The conformation change of Bcl-2 and Bax activation were detected by immunostaining and Western blot analysis. Total expression of Bcl-2 and Bax were examined by Western blot analysis. RESULTS: Arsenic trioxide inhibited the growth of human gastric cancer SGC7901 cells and induced apoptosis. There were two Bcl-2 phenotypes coexisting in SGC7901 cells and the Bcl-2 cytoprotective phenotype could change into a cytodestructive phenotype following conformational change of Bcl-2, triggered by arsenic trioxide exposure. Bax activation might also be involved in arsenic trioxide-induced Bcl-2 conformational change. Arsenic trioxide did not change levels of total Bcl-2 expression, but up-regulated total Bax expression for the treatment time ranging from 3 to 24 hours. CONCLUSION: Arsenic trioxide induces apoptosis through induction of Bcl-2 conformational change, Bax activation and up-regulation of total Bax expression rather than affecting total Bcl-2 expression in human gastric cancer SGC7901 cells. The conformational change of Bcl-2 may be a novel described mechanism of arsenic trioxide-induced apoptosis in cancer cells.

[Effects of matrine on the apoptosis and expression of adhesion molecule in multiple myeloma RMPI8226 cells].

To investigate the effects of matrine on apoptosis and expression of adhesion molecules in human multiple myeloma cell line RPMI8226 cells, RPMI8226 cells were incubated with indicated concentrations of matrine. The growth of RPMI8226 cells was observed by CCK-8 colorimetric assay and apoptosis was detected by flow cytometry using Annexin V-FITC/PI staining. The cell cycles were analyzed by PI staining. Flow cytometry using Annexin V-FITC/PI staining was used to detect the expression of cell adhesion molecules, including CD44, CD44v6, CD54 and CD106. The results showed that RPMI8226 cell viability in presence of matrine decreased markedly in a dose- and time-dependent manners. The apoptosis could be induced by matrine and its level increased following the augmentation of the drug concentration. After treated by matrine for 48 hours, a concentration-dependent increase of cells in G(0)/G(1) phase and a decrease in S phase could be detected, but no obvious change of cell count was found in G(2)/M phase. Treatment of RPMI8226 cells with matrine for 48 hours resulted in decrease of expression levels of CD44 and CD54, while expressions of CD44v6 and CD106 had no significant change. It is concluded that matrine induces in vitro apoptosis, suppresses proliferation in multiple myeloma cells and depresses expression of some adhesion molecules.